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Definitions

• the present invention relates to a herpes viral mutant capable of selectively targeting tumor cells and/or other specific cell populations. More particularly, the present invention relates to the use of cell-specific and/or tumor-specific promoters to retarget mutant herpes viral vectors toward tumors and specific cell types.
• the cell-specific and/or tumor-specific promoter is used to drive expression of the herpes gamma ( ⁇ ) 34.5 gene, whose gene product is responsible for producing large quantities of progeny virus in infected cells.
• Herpes vectors without the ⁇ 34.5 gene do not replicate well, which is desirable for clinical use. However, the absence of the ⁇ 34.5 gene, also diminishes the ability of the virus to kill tumors or any other infected tissue.
• the present invention allows for the production of high amounts of herpes virus in cells that can use the cell- and/or tumor-specific promoter. Cells that cannot turn on the promoter, however, do not support viral replication, thus saving them and their neighboring cells from active and noxious viral infection and replication, thus, redirecting herpes' virulence towards desired target cells.
• Neoplasia is a process that occurs in cancer, by which the normal controlling mechanisms that regulate cell growth and differentiation are impaired, resulting in progressive growth. This impairment of control mechanisms allows a tumor to enlarge and occupy spaces in vital areas of the body. If the tumor invades surrounding tissue and is transported to distant sites (metastases) it will likely result in death of the individual.
• the desired goal of cancer therapy is to kill cancer cells preferentially, without having a deleterious effect on normal cells.
• Several methods have been used in an attempt to reach this goal, including surgery, radiation therapy, and chemotherapy.
• Surgery was the first cancer treatment available, and still plays a major role in diagnosis, staging, and treatment of cancer, and may be the primary treatment for early cancers (see, Slapak, CA. and Kufe, D.W., "Principles of Cancer Therapy," in Harrison 's Principles of Internal Medicine, Fauci, A.S. et al. , eds., 14th Ed., McGraw-Hill Cos., Inc., New York, 1998, at 524).
• Radiation therapy is another local (nonsystemic) form of treatment used for the control of localized cancers. Id. at 525. Many normal cells have a higher capacity for intercellular repair than neoplastic cells, rendering them less sensitive to radiation damage. Radiation therapy relies on this difference between neoplastic and normal cells in susceptibility to damage by radiation, and the ability of normal organs to continue to function well if they are only segmentally damaged. Id. Thus, the success of radiation therapy depends upon the sensitivity of tissue surrounding the tumor to radiation therapy. Id. Radiation therapy is associated with side effects that depend in part upon the site of administration, and include fatigue, local skin reactions, nausea and vomiting. Id. at 526. In addition, radiation therapy is mutagenic, carcinogenic and teratogenic, and may place the patient at risk of developing secondary tumors. Id.
• Local treatments such as radiation therapy and surgery, offer a way of reducing the tumor mass in regions of the body that are accessible through surgical techniques or high doses of radiation therapy.
• more effective local therapies with fewer side effects are needed.
• these treatments are not applicable to the destruction of widely disseminated or circulating tumor cells eventually found in most cancer patients.
• systemic therapies are used.
• Chemotherapy is the main treatment for disseminated, malignant cancers (Slapak, CA. and Kufe, D.W., "Principles of Cancer Therapy,” in Harrison 's Principles of Internal Medicine, Fauci, A.S. etal, eds., 14th Ed., McGraw-Hill Cos., Inc., New York, 1998, 527).
• chemotherapeutic agents are limited in their effectiveness for treating many cancer types, including many common solid tumors. Id. This failure is in part due to the intrinsic or acquired drug resistance of many tumor cells. Id. at
• chemotherapeutic agents Another drawback to the use of chemotherapeutic agents is their severe side effects. Id. at 532. These include bone marrow suppression, nausea, vomiting, hair loss, and ulcerations in the mouth. Id. Clearly, new approaches are needed to enhance the efficiency with which a chemotherapeutic agent can kill malignant tumor cells, while at the same time avoiding systemic toxicity.
• gliomas which are the most common primary malignancy arising in the human brain, defy the current modalities of treatment.
• chemotherapy e.g., chemotherapy, and radiation therapy
• glioblastoma multiforme the most common of the gliomas.
• Kursoenberg in Oncology of the Nervous System, M. D. Walker, ed., Boston, Mass., Martinus Nijhoff (1983); Levin et al. , Chapter 46 in Cancer: Principles and Practice of Oncology, vol. 2, 3rd ed., De Vita et al, eds., Lippincott Press, Philadelphia (1989), pages 1557-1611).
• Gliomas represent nearly 40% of all primary brain tumors, with glioblastoma multiforme constituting the most malignant form (Schoenberg, "The Epidemiology of Nervous System Tumors," in Oncology of the Nervous System, Walker, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1983)).
• the five year survival rate for persons with this high grade type of astrocytoma is less than 5 percent, given the current treatment modalities (surgery, radiation therapy and/or chemotherapy) (Mahaley etal, Neurosurgery 71: 826-836 (1989); Schoenberg, in Oncology of the Nervous System, Walker, ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
• glioblastomas After treatment with radiation therapy, glioblastomas usually recur locally. Hochberg, F.H., etal, Neurology 30:901 '-911 (1980). Neurologic dysfunction and death in an individual with glioblastoma are due to the local growth of the tumor. Systemic metastases are rare. Id. For this reason, regional cancer therapy methods, rather than systemic methods, may be especially suitable for the treatment of glioblastomas. Moreover, glioblastomas are resistant to many chemotherapeutic agents, perhaps due to the proliferative characteristics of this tumor type.
• chemotherapeutic agents are cell-cycle-active, i. e. , cytotoxic primarily to actively cycling cells (Slapak, C.A., and Kufe, D.W., "Principles of Cancer Therapy,” in Harrison 's Principles of Internal Medicine, Fauci, A.S. et al, eds., 14th Ed., McGraw-Hill Cos., Inc., New York, 1998, 527).
• chemotherapy is most effective for cancers with a small tumor burden where the growth fraction of the tumor is maximal. Id.
• the growth fraction for glioblastoma tumors is only 30%, with the remaining 70% of cells being in G 0 , a resting phase (cells in G 0 may die or may re-enter the active cell cycle (Yoshii et al, J. Neurosurg. (£5:659-663 ( 1986)). While the 30% of glioblastoma cells that are actively dividing contribute to the lethal progression of this tumor, the 70% that are quiescent are responsible for the resistance of these tumors to a number of chemotherapeutic agents that target actively proliferating cells.
• Proposed viral cancer therapies include two distinct approaches : ( 1 ) direct cell killing (oncolysis) by a mutagenized virus (Martuza et al. , Science 252:854- 856 (1991); MineXdi etal, Nature Medl :93%-943 (1995); Boviatsis etal, Cancer Res. 54: 5745-5751 (1994); Kesari, et al, Lab. Invest.
• viruses for use as oncolytic agents have initially focused on the use of replication-incompetent viruses. This strategy was hoped to prevent damage to non-tumor cells by the viruses. A major limitation of this approach was that these replication-incompetent viruses required a helper virus to be able to integrate and/or replicate in a host cell.
• One example of the viral oncolysis approach the use of replication-defective retroviruses for treating nervous system tumors, requires the implantation of a producer cell line to spread the virus. These retroviruses are limited in their effectiveness, because each replication-defective retrovirus particle can enter only a single cell and cannot productively infect others thereafter.
• HSV-1 herpes simplex virus type 1
• the first consists of viral mutants with defects in the function of a viral gene needed for nucleic acid metabolism, such as thymidine kinase (Martuza, R.L., et al, Science 252:854-856 (1991)), ribonucleotide reductase (RR) (Goldstein, D.J. & Weller, S.K., J. Virol. -52:196-205 (1988); Boviatsis, E.J., et ⁇ /., Gene Ther. 7:323-331 ( 1994); Boviatsis, E.J., etal, Cancer Res. 54:5145-5151 (1994); Mineta, T., et ⁇ /., Cancer Res. 54:3363-3366 (1994)), or uracil-N-glycosylase (Pyles, R.B. and Thompson, R.I., J. Virol. 68:4963-4912 (1994)).
• the second consists of viral mutants with defects in the function of the ⁇ 34.5 gene (Chambers, R., et al, Proc. Natl Acad. Sci. USA 92:1411-1415 (1995)), which functions as a virulence factor by markedly enhancing the viral burst size of infected cells through suppression of the shutoff of host protein synthesis (Chou, J., etal, Science 250: 1262- 1266 (1990); Chou, J. andRoizman, B., Proc. Nat Acad. Sci. USA 89:3266-3210 (1992)).
• HSV viruses that are multiply mutated have been developed. These include mutants G207 (Mineta, T., et al, Nat. Med. 7:938-943 (1995); U.S. Patent 5,585,096, issued December 17, 1996 to Martuza et al), and MGH1 (Kramm, CM., et al, Hum. Gene Ther. 5:2057-2068 (1997), which possess deletions of both copies of ⁇ 34.5 and an insertional mutation of RR.
• Another multiply mutated HSV virus is the ⁇ 34.5/uracil DNA glycosylase (UNG) mutant strain 3616UB (Pyles, R.B., et al, Hum. Gene Ther. 5:533-544 (1997)).
• UNG uracil DNA glycosylase
• These double mutant strains demonstrate markedly reduced neurovirulence upon direct intracranial injection, retain sensitivity to ganciclovir, and show relatively selective replication in tumor cells compared to normal tissues.
• Such double mutant HSV strains retain the defective ⁇ 34.5 gene, thus demonstrating little virulence towards normal tissues. Although, they clearly demonstrate oncolytic effects against tumor cells, such effects are less than those observed in mutants with intact ⁇ 34.5 genes (Kramm, CM., et al, Hum. Gene Ther. 5:2057-2068 (1997); Qureshi, N. and Chiocca, E.A., unpublished data).
• the toxicity exhibited by an intact ⁇ 34.5 gene might reduce the potential application of the latter viruses as on
• the second approach in viral cancer therapy is the viral delivery of anticancer transgenes, whereby the phenotype of the target tumor cells is genetically altered to increase the tumor's drug sensitivity and responsiveness.
• This approach involves directly transferring a "chemosensitization” or "suicide” gene encoding a prodrug activation enzyme to malignant cells, in order to confer sensitivity to otherwise innocuous agents (Moolten, F.L., Cancer Gene Therapy 7:279-287 (1994); Freeman, S.M., et al, Semin. Oncol. 23:31-45 (1996); Deonarain, M. P., et al, Gene Therapy 2: 235-244 (1995)).
• herpes simplex virus thymidine kinase (HSV-TK) in combination with the prodrug ganciclovir represents a prototypic prodrug/enzyme activation system known in the art with respect to its potential applications in cancer gene therapy.
• HSV-TK phosphorylates the prodrug ganciclovir and generates nucleoside analogs that induce DNA chain termination and cell death in actively dividing cells.
• Tumor cells transduced with HSV-TK acquire sensitivity to ganciclovir, a clinically proven agent originally designed for treatment of viral infections. Moolten, F.L. and Wells, J.M., J. Natl. Cancer lnst.
• the bacterial gene cytosine deaminase is a prodrug/enzyme activation system that has been shown to sensitize tumor cells to the antifungal agent 5-fluorocytosine as a result of its transformation to 5-flurouracil, a known cancer chemotherapeutic agent (Mullen, C.A., et al. , Proc.
• prodrug-activating enzyme systems have also been investigated (T. A. Connors, Gene Ther. 2:702-709 (1995)). These include the bacterial enzyme carboxypeptidase G2, which does not have a mammalian homolog, and can be used to activate certain synthetic mustard prodrugs by cleavage of a glutamic acid moiety to release an active, cytotoxic mustard metabolite (Marais, R., et al, Cancer Res. 56: 4735-4742 (1996)), and E.coli nitro reductase, which activates the prodrug CB 1954 and related mustard prodrug analogs (Drabek, D., et al, Gene Ther. 4:93-100 (1997); Green, N.K., et al,
• CYP cytochrome P450 gene
• P450 cytochrome P450 gene
• a cancer chemotherapeutic agent that is activated through a P450-catalyzed monoxygenase reaction
• the P450-based drug activation strategy utilizes a mammalian drug activation gene (rather than a bacterially or virally derived gene), and also utilizes established chemotherapeutic drugs widely used in cancer therapy.
• cytochrome P450 enzymes to yield metabolites that are cytotoxic or cytostatic toward tumor cells.
• cancer chemotherapeutic drugs such as cyclophosphamide (CPA), its isomer ifosfamide (IF A), dacarbazine, procarbazine, thio-TEPA, etoposide, 2-aminoanthracene, 4-ipomeanol, and tamoxifen (LeBlanc, G.A. and Waxman, D.J., Drug Metab. Rev. 20:395-439 (1989); Ng, S.F. and Waxman D.J., Intl. J. Oncology 2:731-738 (1993); Goeptar, A.R., et al, Cancer Res. 54:2411-2418 (1994); van Maanen, J.M., et al, Cancer Res. 47:4658-4662
• tumor cells were rendered highly sensitive to CPA or IF A by transduction of C YP2B 1 , which encodes a liver P450 enzyme that exhibits a high rate of CPA and IFA activation (Clarke, L. and
• transgenes comprising prodrug-activating or "suicide” genes
• cytokine genes to enhance immune defense against the tumor
• RNA, and ribozymes Zaaia, 3.A., etal, Ann. N. Y. Acad. Sci. 660:95-106 (1992)).
• the ability of the drug to kill tumor cells is limited by the stage of the cell cycle of the cells as GCV targets only cells in the process of DNA replication. It is thus unlikely that therapeutic gene delivery by these replication-defective vectors will affect tumor cells distant from the inoculation site, even in instances where the therapeutic gene produces a freely diffusible anticancer agent, such as cytokines or CPA metabolites.
• the present invention overcomes the disadvantages of the prior art by providing a herpes viral mutant that can selectively target neoplastic cells for viral oncolysis by the transcriptional retargeting of ⁇ 34.5's action.
• the herpes viral mutant of the invention can also target other cell populations as well.
• the inventors reintroduced the ⁇ 34.5 gene into a RR/ ⁇ 34.5 double mutant strain (MGH1) under the transcriptional control of the cell-cycle regulated, cellular B-myb promoter. They demonstrated that this novel oncolytic virus (called "Myb34.5”) remained as oncolytic as a single RR mutant virus that possessed a wild-type ⁇ 34.5 gene, yet retained a favorable toxicity profile similar to that of a ⁇ 34.5 -deletion mutant. These findings thus show that the transcriptional retargeting of a viral gene responsible for preventing the shutoff of protein synthesis of infected cells provides an avenue for achieving selective oncolysis.
• MGH1 RR/ ⁇ 34.5 double mutant strain
• the transcriptional retargeting of ⁇ 34.5's expression provides a means to achieve selective virulence for tumors, or other targeted cell populations.
• the herpes viral mutant comprises a deletion or inactivating mutation in both copies of the gene encoding ⁇ 34.5, wherein at least one copy of the ⁇ 34.5 gene is reintroduced under the transcriptional control of a cell-specific and/or tumor-specific promoter.
• herpes viral gene there may be more than one specific endogenous deletion or inactivating mutation of a herpes viral gene, in addition to the ⁇ 34.5 gene.
• the gene encoding RR encodes the small subunit (UL40).
• Any other herpes viral genes may also be deleted, such as, e.g. , thymidine kinase (TK), uracil DNA glycosylase (UNG), or dUTPase.
• TK thymidine kinase
• UNG uracil DNA glycosylase
• dUTPase dUTPase.
• the herpes mutant of the invention is also capable of delivering a transgene whose product could be cytotoxic to tumor cells.
• the transgene could encode a product capable of activating or enhancing a chemotherapeutic agent (e.g., a suicide gene, such as HSV-TK, CD, or cytochrome P450).
• a chemotherapeutic agent e.g., a suicide gene, such as HSV-TK, CD, or cytochrome P450.
• the transgene can be a cytokine gene to enhance tumor immunogenicity (e.g, tumor necrosis factor alpha (TNF- ⁇ ), interleukins (IL-2, IL-4), interferon- ⁇ , granulocyte-macrophage colony stimulating factor
• the herpes mutant further comprises a transgene encoding a gene product capable of converting a chemotherapeutic agent to its cytotoxic form, a cytokine gene, or any other tumoricidal transgene.
• the transgene can be inserted in the original ⁇ 34.5 deletion or anywhere in the UL40 locus.
• the invention provides a preferred embodiment of the foregoing herpes viral mutant where the transgene encodes a suicide gene that activates a chemotherapeutic agent.
• a particularly preferred example of such a suicide gene is mammalian cytochrome P450. More particularly, this cytochrome P450 may be P450 2B1, or alternatively P450 2B6, P450 2A6, P450 2C6, P450 2C8, P450 2C9, P450 2C11, or P450 3A4. P450 2B1 is particularly preferred. If such a suicide gene is present in the foregoing viral mutant, then the chemotherapeutic agent that would be activated is a member of the oxazosphorine class.
• the agent would be cyclophosphamide, ifosfamide, N-methyl cyclophosphamide, methylchloropropylnitrosourea, polymeric cyclophosphamide, polymeric ifosfamide, polymeric N-methyl cyclophosphamide, or polymeric methylchloropropylnitrosourea.
• the invention also provides an embodiment of the foregoing herpes viral mutants, wherein the herpes mutant is a herpes simplex virus, and more particularly, wherein the mutant is herpes simplex virus (HSV) type 1 or type 2. HSV- 1 is particularly preferred.
• HSV herpes simplex virus
• the herpes viral mutant is derived from HSV-1 , and comprises: (a) a deletion or inactivating mutation in both copies of the gene encoding ⁇ 34.5; and (b) an insertion of at least one copy of said ⁇ 34.5 gene under transcriptional control of a cell-type specific and/or tumor specific promoter, such that said promoter drives expression of the ⁇ 34.5 gene.
• herpes viral mutants may also be present in the herpes viral mutant of the invention.
• Deletions in herpes RR, TK, UNG, or dUTPase are exemplary herpes viral genes.
• the cell-specific promoter or tumor-specific promoter can be any one of the well-characterized regulatory elements controlling tumor- type and/or cell-type specific gene expression. For a review, see, Miller, N. and Whelan, J., Hum. Gene Ther. 5:803-815 (1997); Walther, W. and Stein, U., J. Mol. Med. 74:379-392 (1996); Schnierie, B.S. and Groner, B., Gene Therapy
• tumor-specific promoters include, e.g., DF3 (MUC 1 ) (which is overexpressed in the majority of breast cancers)(Abe, M. and
• the tumor- specific promoter is B-myb.
• the viral mutant is Myb34.5.
• Exemplary cell-specific promoters include the following: vascular endothelial growth factor (VEGF) receptor (flkl) promoter expressed in endothelial cells (Kappel et al. Blood 93: 4282-4292 (1999); insulin promoter expressed in beta cells of the pancreas (Ray et al, J. Surg. Res. 84: 199-203 (1999); gonadotropin-releasing hormone receptor gene expressed in cells of the hypothalamus (Albarracin et al, Endocrinology 140: 2415-2421 (1999); matrix metalloproteinase 9 promoter, expressed in osteoclasts and keratinocytes (Munant et al, J. Biol. Chem.
• VEGF vascular endothelial growth factor
• flkl vascular endothelial growth factor receptor
• the present invention also provides a method for selectively killing neoplastic cells that overexpresses a known tumor-specific promoter using the herpes viral mutants described above, comprising: infecting said neoplastic cells with said herpes viral mutant, said viral mutant comprising: (a) a deletion or inactivating mutation in a gene encoding ⁇ 34.5; and (b) an insertion of at least one copy of said ⁇ 34.5 gene under the transcriptional control of said tumor specific promoter, such that said promoter drives expression of said ⁇ 34.5 gene; and selectively killing said neoplastic cells.
• tumor-specific promoters include: DF3 (MUC1) (which is overexpressed in the majority of breast cancers)(Abe, M. and Kufe, D., Proc. Natl. Acad. Sci. USA 90:282-286 (1993); Manome, Y., et al, Gene Ther. 2:685, A051 (1995); Chen, L., et al, J. Clin. Invest. 96:2775-2782
• AFP which is overexpressed in hepatoma
• CEA which is overexpressed in colon and lung cancers
• Thompson J.A., et al, J. Clin. Lab. Anal. 5:344-366 (1991); Osaki, T., et al, Cancer Res. 54:5258-5261 (1994)
• PSA which is overexpressed in prostate cancers
• the tumor-specific promoter is B-myb.
• the viral mutant is Myb34.5.
• deletions or mutations in other herpes viral genes may also be present in the herpes viral mutant used in the method of the invention.
• Deletions in herpes RR, TK, UNG, or dUTPase are exemplary herpes viral genes.
• the invention provides the above method for selectively killing neoplastic cells, wherein said herpes viral mutant further comprises a transgene, wherein the transgene is a suicide gene, a cytokine gene, or any tumoricidal gene.
• the method further comprises contacting the neoplastic cells with a chemotherapeutic agent capable of being activated by said suicide gene and selectively killing the neoplastic cells.
• a chemotherapeutic agent capable of being activated by said suicide gene and selectively killing the neoplastic cells.
• the preferred suicide gene is cytochrome P450.
• P4502B1 is particularly preferred.
• the cytochrome P450 encoded is P450 2B6, P450 2A6, P450 2C6, P450 2C8, P450
• the chemotherapeutic agent is preferably a member of the oxazosphorine class, particularly cyclophosphamide, ifosfamide, N-methyl cyclophosphamide, methylchloropropylnitrosourea, polymeric cyclophosphamide, polymeric ifosfamide, polymeric N-methyl cyclophosphamide, or polymeric methylchloropropylnitrosourea.
• Another embodiment of the present invention is a method for selectively eliminating a target cell population that overexpresses a known cell-specific promoter using the herpes viral mutants of the invention, comprising: infecting said target cells with said herpes viral mutant, said viral mutant comprising: (a) a deletion or inactivating mutation in a gene encoding ⁇ 34.5; and (b) an insertion of at least one copy of said ⁇ 34.5 gene under the transcriptional control of said cell-specific promoter, such that said promoter drives expression of said ⁇ 34.5 gene; and selectively eliminating a target cell population.
• Exemplary cell-specific promoters include the following: vascular endothelial growth factor (VEGF) receptor (flkl) promoter expressed in endothelial cells (Kappel et al. Blood 93: 4282-4292 (1999); insulin promoter expressed in beta cells of the pancreas (Ray et al, J. Surg. Res. 84: 199-203 (1999); gonadotropin-releasing hormone receptor gene expressed in cells of the hypothalamus (Albarracin et al, Endocrinology 140: 2415-2421 (1999); matrix metalloproteinase 9 promoter, expressed in osteoclasts and keratinocytes (Munant et al, J. Biol. Chem.
• VEGF vascular endothelial growth factor
• flkl vascular endothelial growth factor receptor
• herpes viral mutant used in the method of the invention.
• Deletions in herpes RR, TK, UNG, or dUTPase are exemplary herpes viral genes. Examplary applications of this embodiment include the following:
• Treatment options to eliminate a noxious cell population For example, in conditions where there is exuberant neovascularization of blood vessels, such as cerebral Moya-Moya disease, use of the flkl receptor promoter to drive gamma 34.5 gene expression would allow for selective elimination of the blood vessels causing this disease. Another example is in conditions where there is extensive bone remodeling and elimination of bone, such as osteoporosis, the use of the matrix metalloproteinase 9 or the parathyroid hormone receptor to drive expression of gamma 34.5 would eliminate bone osteoclasts from further remodeling of bone.
• Another embodiment of the invention is a pharmaceutical composition containing any of the foregoing viral mutants, wherein this composition may also contain one or more pharmaceutically acceptable excipients.
• FIG. IA depicts schematic maps of the HSV strains F (wild-type), MGH1 (double RR(ICP6)/ ⁇ 34.5 mutant), and Myb34.5. All strains contain the typical HSV genome with two unique segments, UL and US, each flanked by inverted repeat elements, ab and ca, respectively (McGeoch, D.J., et al, J. Gen . Virol. 72:3057-3075 (1991)). Depending on their localization in either unique or repeat segments, the HSV genes are present in one or two copies.
• the black box indicates the lacZ insertion into a BamHI site within ICP6 (Goldstein, D.J. & Weller, S.K., J. Virol. 62:2970-2977 (1988)), while ⁇ indicates the deletions within ⁇ 34.5 in MGH1 and Myb34.5 (Kramm, CM., et al, Hum.
• the hatched bar indicates recombination of the B-myb promoter- ⁇ 34.5 construct into the ICP6 locus.
• Fig. IB depicts characterization of the HSV mutant Myb34.5 by Southern blot analysis.
• Hybridization of Xhol digested viral DNA to a full-length probe for ICP6 reveals the expected 9.0 kB fragment sizes for the ICP6 gene with a full length lacZ insertion in MHG1 (lane 2) and MybRevt (lane 4).
• Myb 34.5 (lane 3), there is replacement by the B-myb promoter/ ⁇ 34.5 cassette and further deletion of ICP6 to give a 6.7 kB band.
• DNA from wild-type F strain is in lane 1.
• Fig. IC depicts hybridization with a lacZ probe, and reveals fragments with MGH1 (lane 2) and MybRevt (lane 4), with no hybridization to Myb34.5 (lane 3).
• Fig. ID depicts a BstEII-Bbs fragment of ⁇ 34.5, internal to the deleted regions of R3616 and MGH 1 , which reveals a 5.3 kB fragment in BamHI digested Myb34.5 (DNA (lane 3), and several bands in F (lane 1), but fails to hybridize to either MGH1 (lane 2) or MybRevt (lane 4).
• Figure 2 depicts an autoradiographic image of electrophoretically separated lysates of infected cells demonstrating inhibition of host protein synthesis shutoff by mutant viral strains.
• Human neuroblastoma cells (SK-N-SH) were plated at 1 x 10 6 cells/100mm dish. Twenty-four hours later cells were infected at an MOI of 3.0.
• Figure 3 is a bar graph depicting viral replication in arrested and cycling cells.
• Human embryo-derived primary fibroblasts (CRL 7706) were plated at 1 x 10 5 cells/60 mm dish. Forty-eight hours after plating, media was replaced with DMEM containing 20 ⁇ M Lovastatin for 36 hours (hatched bars). Triplicate plates were counted and infected at a multiplicity of infection (MOI) of 1.0 with various mutant strains (triplicate experiments). Forty-eight hours after infection, cells and supernatants were harvested and virus liberated by freeze -thaw cycles. Parallel experiments were performed with cells allowed to remain in medium containing 10% fetal bovine serum (solid bars).
• Figures 4A and 4B depict in vivo growth inhibition by Myb34.5.
• rat gliosarcoma 9L Fig.4A
• human U87 ⁇ EGFR glioma Fig. 4B
• vehicle or mutant viral strains were inoculated intratumorally into tumors.
• Arrows indicate the times of viral injection (days 1, 3, 5, 7), while values are the averages of five mice per group (9L) and six per group (U87 ⁇ EGFR).
• the present invention relates to the selective killing of neoplastic cells by viral mediated oncolysis alone or the combination of viral mediated oncolysis and suicide gene therapy.
• the invention provides for a herpes viral mutant, a method of selectively killing neoplastic cells using this herpes viral mutant, and a pharmaceutical composition containing the viral mutant.
• the invention also provides a method for selectively eliminating target cell populations using the herpes viral mutant of the invention.
• the invention provides for a herpes viral mutant, wherein the mutant is genetically engineered to have (a) a deletion or inactivating mutation in both copies of the gene encoding ⁇ 34.5; and (b) an insertion of at least one copy of said ⁇ 34.5 gene under the transcriptional control of a tumor-specific or cell-type specific promoter, such that said promoter drives expression of the ⁇ 34.5 gene.
• the herpes mutant may also contain one or more additional deletions or mutations in other herpes viral genes, such as, e.g. , RR, TK, UNG, and dUTPase.
• the herpes viral mutant can also deliver a transgene encoding a product that activates a chemotherapeutic agent, a cytokine gene, or any other tumoricidal gene.
• herpes viral mutants of the invention may be derived from several different types of herpes viruses.
• Herpes viruses that may be used to derive the viral mutants of the invention include herpes simplex virus (HSV), cytomegalovirus, Epstein-Barr virus, varicella zoster virus, and pseudorabies virus.
• HSV herpes simplex virus
• cytomegalovirus Epstein-Barr virus
• varicella zoster virus varicella zoster virus
• pseudorabies virus pseudorabies virus.
• Herpes simplex viruses are of particular interest.
• herpes simplex virus any member of the subfamily herpesviridae alpha containing a deletion or inactivating mutation as described above.
• a preferred embodiment of the invention employs HSV-1 or HSV-2 to create the herpes viral mutant, with
• HSV-1 being the most preferred.
• HSV-1 is a human neurotropic virus that is capable of infecting virtually all vertebrate cells. Natural infections follow either a lytic, replicative cycle or establish latency, usually in peripheral ganglia, where the DNA is maintained indefinitely in an episomal state. HSV-1 contains a double-stranded, linear DNA genome, 153 kilobases in length, which has been completely sequenced by
• herpes ⁇ 34.5 gene precludes the host cell's response to viral infection, namely the triggering of host protein synthesis shutoff in an apoptotic-like response
• apoptotic-like response Chou, J., et al, Science 250:1262-1266 (1990); Chou, J. and Roizman, B., Proc. Natl. Acad. Sci. USA 59:3266-3270 (1992); Chou, j., etal, Proc. Natl. Acad. Sci. USA 92:10516-10520 (1995)).
• a similar function is widespread among pathogenic viruses (Cosentino, G.P., et al, Proc. Natl. Acad. Sci. USA 92:9445-9449 (1995);
• ⁇ 34.5 is nonessential for viral growth in culture in Vero cells, it enables the virus to spread in the central nervous system (CNS) of mice, and maps to a region of the HSV genome previously implicated in CNS replication (Markovitz, N.S., et al, J. Virol. 77:5560-5569 (1997); Centifanto-Fitzgerald, Y.M., et al, J. Esp. Med. 755:475-489 (1982)). This may be due to the fact that the ⁇ 34.5-encoded protein inhibits the double-stranded RNA-dependent kinase (PKR).
• PSR RNA-dependent kinase
• PKR phosphorylates the alpha subunit of elongation initiation factor eIF-2, resulting in inhibition of protein synthesis (Chou, J., et al, Science 250:1262-1266 (1990); Chou, J. and Roizman, B., Proc. Natl. Acad. Sci. USA 59:3266-3270 (1992); Chou, J., et al, J. Virol 65:8304-8311 (1994)). Infection of cells of neuronal origin with mutants incapable of expressing ⁇ 34.5 results in shut-off of cellular protein synthesis, with the resultant limitation of viral production.
• HSV in the presence of ⁇ 34.5, HSV will prevent apoptosis, thus allowing for production of progeny viruses. In its absence, the cell dies and the infecting HSV cannot generate progeny viruses. Thus, HSV infection/propagation throughout an organ is eliminated.
• the tumor- or cell type-specific promoter/ ⁇ 34.5 approach of the invention thus allows for production of virus in cells that can use that promoter, but cells that cannot turn on the promoter will not propagate infection.
• the herpes viral mutant of the invention comprises a deletion or inactivating mutation in both copies of the ⁇ 34.5 gene, wherein at least one copy of the ⁇ 34.5 gene is reintroduced under the control of a cell-specific or tumor-specific promoter.
• deletion is intended to mean the elimination of nucleic acids from a gene, such as the ⁇ 34.5 gene.
• activating mutation is intended to broadly mean a mutation or alteration to a gene wherein the expression of that gene is significantly decreased, or wherein the gene product is rendered nonfunctional, or its ability to function is significantly decreased.
• gene encompasses both the regions coding the gene product as well as regulatory regions for that gene, such as a promoter or enhancer, unless otherwise indicated.
• Ways to achieve such alterations include: (a) any method to disrupt the expression of the product of the gene or (b) any method to render the expressed gene nonfunctional.
• Numerous methods to disrupt the expression of a gene are known, including the alterations of the coding region of the gene, or its promoter sequence, by insertions, deletions and/or base changes. (See, Roizman, B. and Jenkins, F.J., Science 229: 1208-1214 (1985)).
• a cell-specific and/or tumor-specific promoter is used to drive expression of ⁇ 34.5.
• the promoter can be any one of the well-characterized regulatory elements controlling tumor-type and/or cell-type specific gene expression.
• Miller, N. and Whelan, J., Hum. Gene Ther. 5:803-815 (1997); Walther, W. and Stein, U., J. Mol Med. 74:319-392 (1996); Schnierie, B.S. and Groner, B., Gene Therapy
• promoter is intended to mean the DNA region, usually upstream to the coding sequence of a gene or operon, which binds RNA polymerase and directs the enzyme to the correct transcriptional start site.
• cell-specific promoter is intended a promoter that directs expression in particular cell types.
• a “tumor-specific” promoter can also be considered a “cell-specific” promoter (i.e., it is specific for tumor cells).
• a “cell-specific promoter”, as used herein, is intended to exclude tumor-specific promoters, unless otherwise indicated.
• Exemplary cell-specific promoters include the following: vascular endothelial growth factor (VEGF) receptor (flkl) promoter expressed in endothelial cells (Kappel et al. Blood 93: 4282-4292 (1999); insulin promoter expressed in beta cells of the pancreas (Ray et al, J. Surg. Res.
• Treatment options to eliminate a noxious cell population In one example, in conditions where there is exuberant neovascularization of blood vessels, such as cerebral Moya-Moya disease, the use of the flkl receptor promoter to drive gamma 34.5 gene expression would allow for selective elimination of the blood vessels causing this disease.
• the use of the matrix metalloproteinase 9 or the parathyroid hormone receptor to drive expression of gamma 34.5 would eliminate bone osteoclasts from further remodeling of bone.
• the dopamine-beta-hydroxylase promoter to eliminate the noradrenergic neurons and then study the effect on animal development.
• tumor-specific promoter is intended a promoter that is induced selectively or expressed at a higher level in the target tumor cell than in a normal cell .
• the tumor targeting specificity for the herpes viral mutant of the invention is achieved by the use of tumor-specific promoters to selectively activate expression of the transduced gene in the tumor cell at either the primary tumor site or its metastases (Miller, N. and Whelan, J., Hum. Gene Ther. 5:803-815 (1997); Walther, W. and Stein, U., J. Mol. Med. 74:379-392 (1996); Schnierie, B.S. and Groner, B., Gene Therapy 3:1069-1073 (1996); Lan, K-H., et al, Cancer Res. 57:4279-4284 (1997); Dachs, G.U., et al, Oncol. Res. 9:313-325 (1997)).
• tumor-specific promoters include those that have been derived from genes that encode tyrosinase (allowing for targeting to melanoma) (Vile, R.G. and Hart, I.R., Cancer Res. 53:962-967 (1993); Vile, R.G. and Hart, I.R., Ann. Oncol 5 (Suppl. 4 :S59-S65 (1994); Hart, I.R., et al, Curr. Opin. Oncol. 6:221-225 (1994)); c-erbB-2 oncogene (targeting to breast, pancreatic, gastric, and ovarian cancers) (Hollywood, D., and Hurst, H., EMBO J 72:2369-
• CEA carcinoembryonic antigen
• Tumor cells are also known to overexpress particular oncogenes, so that cells with upregulated gene expression can be targeted using promoter elements of such genes.
• B-myb, C-myb, c-myc, c-kit, and the c-erbB2 oncogene are some representative examples of these types.
• the B-myb promoter (see, Lyon, J., et al, Crit. Rev.Oncogenesis 5:373-388 (1994) contains a consensus E2F binding site, is strictly regulated in cycling cells, and is in fact repressed in G 0 (Lam, E.W. and Watson, R.J., EMBO J.
• the B-myb promoter is a particularly preferred tumor-specific promoter.
• Any cancer type having a well-characterized promoter would find use in the invention.
• Examples of such promoters can be found in Table 1 of Clary, B.M., et al, Cancer Gene Therapy 7:565-574 (1998); Table I of Spear, M.A., Anticancer Research 75:3223-3232(1998); Table 2 of Walther, W. and Stein, U.,
• the viral mutants of the invention may also possess additional mutations in any viral gene(s), but most preferably in a gene required for replication, whose mammalian homologue is up-regulated by elevated levels of E2F.
• mammalian ribonucleotide reductase (m7?i?) is up-regulated during the G, phase of the cell cycle and its transcription is regulated by "free"
• Elevation in the levels of free E2F causes increased expression of several mammalian genes whose viral homologues are required for replication of the virus.
• these genes include thymidine kinase (TK), uracyl DNA glycosylase (UNG), and uracyl-triphosphatase enzymes (dUTPase).
• TK thymidine kinase
• UNG uracyl DNA glycosylase
• dUTPase uracyl-triphosphatase enzymes
• Viruses containing a mutation in one or more of these genes would replicate selectively in cells with elevated levels of free E2F.
• the invention encompasses herpes viral mutants having a mutation in one or more of these genes, in addition to the mutation in ⁇ 34.5.
• the mutation is in a ribonucleotide reductase gene.
• E2F (including E2F 1 , E2F2, E2F3 , E2F4, E2F5) appears to be the primary mediator of the cell cycle-regulated transcriptional cascade that involves pi 6, cyclin D/ cdk4, and pRB (DeGregori et al, Mol. Cell. Biol. 15: 4215-4224 (1995); Lukas et ⁇ /., Mol. Cell. Biol. 16: 1047-1057 (1996; Oyr ⁇ ac tet L, Genes Dev. 8: 1772-1786 (1994)).
• defects in a gene involved in this cascade can lead to increased levels of E2F and thereby increased levels of mammalian RR,
• TK RNA kinase 4
• Cdk6 cyclin D kinase 6
• DP 1 , DP2, and/or DP3 may also lead to increased liberation of E2F.
• RR " , TK “ , UNG “ and dUTPase ' viral mutants may effectively target a large percentage of tumor cells, particularly if they possess a defect in the pi 61 cyclin D/ p7?73 pathway that leads to an increase in "free" E2F.
• tumor cells from many different origins possess alterations in the pathways described above leading to elevated levels of RR, TK, UNG and dUTPase, and thus are targets for the viral mutant of the invention.
• the glioma tumor cell lines (rat 9L, human U87, and human T98 cells) possess inactivating mutations of pi 6 (Van Meir et al, Cancer Res. 54: 649-652 (1994)), as well as elevated levels of mRR. These cells were thus able to complement the replication of the HSV-1 derived viral mutant rRp450 to levels close to that of the wild-type
• ribonucleotide reductase gene is intended a nucleic acid that encodes any subunit or part of the enzyme, ribonucleotide reductase, such that when this nucleic acid is expressed in a cell, this part or subunit is produced, whether functional or nonfunctional.
• Ribonucleotide reductase (RR) is a key enzyme in the de novo synthesis of DNA precursors, catalyzing the reduction of ribonucleotides to deoxyribonucleotides.
• HSV-1 encodes its own RR (UL39 and UL40 genes), which is composed of two non- identical subunits (Duita, J. Gen. Virol. 64: 513 (1983)).
• the large subunit (140k molecular weight), designated ICP6, is tightly associated with the small subunit (38k molecular weight).
• Herpes simplex virus RR has been found to be required for efficient viral growth in non-dividing cells but not in many dividing cells (Goldstein and Weller, J. Virol. 62: 196 (1988); Goldstein and Weller, Virol. 166: 41 (1988); Jacobson et al, Virol. 173: 276 ( 1989)). Mutations in the small subunit of RR also lead to loss of RR activity and neuropathogenicity (Cameron et al, J. Gen. Virol. 69: 2607 (1988)), however, particularly preferred are mutations in the large subunit.
• the promoter region of ribonucleotide reductase ICP6 has been mapped to the 5' upstream sequences of the ICP6 structural gene (Goldstein and Weller, J. Virol 62: 196 (1988); Sze and Herman, Virus Res. 26: 141 (1992)).
• the transcription start site for the small subunit of RR, namely UL40, falls within the coding region of ICP6 (McLauchlan and Clements, J. Gen. Virol. 64: 997 (1983); McGeoch et al, J. Gen. Virol. 69: 1531 (1988)).
• Viral mutants derived from HSV-2 based on the viral mutants illustrated herein using the HSV-1 genome are encompassed by the present invention.
• HSV-2 contains both RR subunits; moreover, HSV-2 ICP10 is analogous to HSV-1 ICP6.
• RR ribonucleotide reductase deficient
• TK HSV-1 mutants known in the art are resistant to these anti- viral agents, such mutants could be difficult to eliminate in the event of systemic infection or encephalitis.
• TK + viral mutants such as RR ' -HSV mutants, are responsive to antiviral therapy.
• RR ' -HSV mutants are compromised in their ability to produce infections and synthesize viral DNA at 39.5 °C in vitro (Goldstein and Weller, Virology 766:41 (1988)). Therefore, these mutants are attenuated for neurovirulence and less likely to propagate in the event of a fever in the infected host. Such characteristics are important to a therapeutic vector that must be of attenuated neurovirulence and amenable to antiviral therapy in the event of viral encephalitis.
• RR " viral mutants demonstrate another advantage of the viral mutant of the invention.
• a number of host factors could inhibit propagation of the viral mutant.
• treatment with a chemotherapeutic agent and activation by the transgene would provide a supplemental anti-cancer treatment.
• mutation refers to any alteration to a gene wherein the expression of that gene is significantly decreased, or wherein the gene product is rendered nonfunctional, or its ability to function is significantly decreased.
• the term "gene” encompasses both the regions coding the gene product as well as regulatory regions for that gene, such as a promoter or enhancer. Such alterations render the product of the gene non-functional or reduce the expression of the gene such that the viral mutant has the properties of the instant invention. Moreover, the invention encompasses mutants with one or more mutation(s) in one or more gene(s) of interest. Thus, by “a” is intended one or more. Ways to achieve such alterations include: (a) any method to disrupt the expression of the product of the gene; or (b) any method to render the expressed protein nonfunctional.
• HSV-1 mutants The construction of HSV-1 mutants is described, for example, in Martuza et al, U.S. Patent 5,585,096 (Dec. 1996); Roizman et al, U.S. Patent 5,288,641 (Feb. 1994); Roizman, B. and Jenkins, F.J., Science 229:1208-1214 (1985);
• Genetic alterations of the viral genome can be determined by standard methods such as Southern blot hybridization of restriction endonuclease digested viral DNA, sequencing of mutated regions of viral DNA, detection of new (or lost) restriction endonuclease sites, enzymatic assay for ribonucleotide reductase activity (Huszar, D. and Bacchetti, S., J. Virol. 37:580-598 (1981)).
• genetic alteration of the viral genome can be determined by (1) Western blot or ELISA analysis of infected cell proteins with antibodies the viral homologue that has been mutated, e.g. , RR, or (2) Northern blot analysis of infected cells for transcription of the viral homologue that has been mutated, e.g., RR (Jacobson, J.G., et al,
• a viral mutant that has been mutated in one or more genes can be isolated after mutagenesis or constructed via recombination between the viral genome and genetically-engineered sequences.
• up-regulated is intended that expression of the gene(s) encoding the gene product said to be up-regulated is greater than the basal level of expression of this product as found in non-neoplastic cells.
• level of free E2F is elevated is meant that the amount of unbound E2F available in a cell is greater than the amount typically found in non-neoplastic cells.
• selectively killing neoplastic cells is meant that the herpes viral mutant of the invention primarily targets neoplastic cells, rather than non- neoplastic cells.
• neoplastic cells cells whose normal growth control mechanisms are disrupted (typically by accumulated genetic mutations), thereby providing potential for uncontrolled proliferation.
• neoplastic cells can include both dividing and non-dividing cells.
• neoplastic cells include cells of tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas, and the like.
• central nervous system tumors especially brain tumors. These include glioblastomas, astrocytomas, oligodendrogliomas, meningiomas, neurofibromas, ependymomas, Schwannomas, neurofibrosarcomas, etc.
• the invention can be utilized to target for oncolysis both benign and malignant neoplastic cells in the periphery and the brain.
• periphery is intended to mean all other parts of the body outside of the brain.
• a peripheral tumor is intended to mean a tumor in a part of the body outside of the brain.
• the viral mutants of the present invention can also carry a heterologous transgene.
• the transgene can be a suicide gene, that is, a gene that encodes a gene product capable of activating a chemotherapeutic agent to its cytotoxic form, such as HSV-TK, CD, or cytochrome P450.
• the suicide gene is a cytochrome P450 gene.
• gene product capable of converting a chemotherapeutic agent to its cytotoxic form is meant a gene product that acts upon the chemotherapeutic agent to render it more cytotoxic than it was before the gene product acted upon it. Other proteins or factors may be required, in addition to this gene product, in order to convert the chemotherapeutic agent to its most cytotoxic form.
• transgene encoding a gene product capable of converting a chemotherapeutic agent to its cytotoxic form is meant a nucleic acid that upon expression provides this gene product.
• “Cytotoxic” is used herein to mean causing or leading to cell death.
• “Gene product” broadly refers to proteins encoded by the particular gene.
• “Chemotherapeutic agent” refers to an agent that can be used in the treatment of neoplasms, and that is capable of being activated from a prodrug to a cytotoxic form.
• the chemotherapeutic agents for use in the invention do not significantly inhibit replication of the viral mutant, which means that viral replication can occur at a level sufficient to lead to death of the infected cell and to propagate the spread of the virus to other cells.
• cytochrome P450 means a mammalian cytochrome P450 gene such as, P450 2B1, P450 2B6, P450 2A6, P450 2C6, P450 2C8, P450 2C9, P450 2C 11, or P450 3A4. Each of these genes has been linked to activation of the anticancer drugs cyclophosphamide and ifosfamide
• cytochrome P450 can also activate N-methyl cyclophosphamide (N-methyl CPA), methylchloropropylnitrosourea (MCPNU), and polymeric forms of CPA, ifosfamide, N-methyl CPA, and MCPNU.
• N-methyl CPA N-methyl cyclophosphamide
• MCPNU methylchloropropylnitrosourea
• Polymeric forms of chemotherapeutic agents are discussed in Brem, Biomaterials, 77: 699-701 (1990); Buahin and Brem, J. Neurooncol 26: 103-110
• cytochrome P450 family of enzymes include cytochrome P450 family of enzymes and drug-metabolizing cytochrome P450 genes from other species (e.g., mouse, rabbit, hamster, dog, etc.) that are homologous to cytochromes P450 2B1, P450 2B6, P450 2A6, P450 2C6, P450 2C8, P450 2C9, P450 2C 11, or P450 3A4, and whose cDNA sequences are known (Nelson et al, DNA and Cell Biology 72:1-51 (1993)).
• the gene encoding cytochrome P450 2BI is used.
• the chemotherapeutic agent that is activated by the suicide gene should not significantly inhibit replication of the viral mutant so as to allow the viral mutant to kill tumor cells by viral oncolysis, as well as by delivery of the suicide gene.
• the use of a chemotherapeutic agent/transgene combination in which the chemotherapeutic agent, or its active metabolites, act instead by crosslinking DNA or by inhibiting DNA repair would not significantly inhibit replication of the viral mutant.
• chemotherapeutic agent/transgene combinations are encompassed by the viral mutant and methods of the present invention.
• a preferred chemotherapeutic agent/transgene combination is cytochrome P450 combined with CPA, ifosfamide, N-methyl cyclophosphamide, MCPNU, or polymeric forms of: CPA, ifosfamide, N-methyl cyclophosphamide and MCPNU.
• a more preferred chemotherapeutic agent/transgene combination is CPA/cytochrome P450 2B1.
• chemotherapeutic agent/transgene combinations for use in the present invention include: CB1954/E. coli nitroreductase (Friedlos et al, Gene Ther. 5: 105-112 (1998); Green et al, Cancer Gene Ther. 4: 229-238 (1997)); topoisomerase I or II inhibitors/enzyme with esterase-like activity, such as, e.g. ,
• CPT-11/carboxylesterase (Jansenet ⁇ /., Int. J. Cancer 70: 335-340 (1997); Danks et al, Cancer Res. 58: 20-22 (1998)); 4-ipomeanol/cytochrome P450 4B1 (Verschoyle et al, Toxicol Appl. Pharmacol. 123: 193-198 (1993)); and 2- aminoanthracene/cytochrome P4504B1 (Smith et al, Biochem. Pharmacol. 50: 1567-1575 (1995)).
• CPA alkylating agent
• PM phosphoramide mustard
• DNA may be spared from extensive damage and may be thus be repaired more readily than cellular DNA.
• Ganciclovir is one example of a chemotherapeutic agent that, when activated, inhibits viral replication. Although it has been demonstrated that the combination of hrR3 and ganciclovir provides a significant anticancer effect due to the conversion of ganciclovir by the viral thymidine kinase gene (Boviatsis et al, Cancer Res. 54: 5745-5751 (1994)), the converted ganciclovir molecules also inhibit viral replication. For this reason, use of TK/GCV may not be a preferred selection in this paradigm. Prodrug-activating enzymes, such as HSV-TK generate anticancer metabolites that act as "false" nucleotides, producing premature termination of replicating DNA strands.
• these prodrug- activating enzymes would be expected to affect both viral and genomic DNA synthesis and would not be a good choice for use in the herpes viral mutants of the invention that contain a suicide gene.
• Another advantage of using chemotherapeutic agents whose mechanism of action is the cross-linking of DNA or the inhibition of DNA repair enzymes is that these agents are effective against even cells in G 0 .
• the targeted cells do not have to be actively dividing at the time that the drug is administered. This is a significant benefit for tumors in which a large percentage of cells are in G 0 .
• glioblastoma the growth fraction, or the relative proportion of cells proliferating in the tumor at any one time, is only 30%, with the remaining 70% of cells being in G 0 .
• These tumors are especially resistant to chemotherapeutic agents that target only actively dividing cells because, while the 30% of glioblastoma cells that are actively dividing contribute to the lethal progression of this tumor, 70% of the cells are in G 0 and may die or may re-enter the active cell cycle, Yoshii et al, J. Neurosurg. 65:659-663 (1986)).
• the 70% that are quiescent are responsible for the resistance of these tumors to chemotherapeutic agents that target actively proliferating cells.
• the viral mutant and method of the present invention provide an advantage over therapies based on replication-conditional or replication-incompetent viral mediated oncolysis alone, in that those therapies will target only those cells that can complement the viral mutation.
• the viral mutant of the invention targets cells with elevated levels of E2F (primarily neoplastic cells) for replication in, and lysis, expression of the transgene and activation the chemotherapeutic agent provides active metabolites that can then diffuse to surrounding tumor cells. These metabolites can thereby kill even those surrounding tumor cells in G 0 (70% of the cells in a glioblastoma).
• the invention finds particular use in the treatment of glioblastomas.
• the glioblastoma represents approximately 30% or 50%) of all primary brain tumors and, despite surgery, chemotherapy, and radiation therapy, is almost universally fatal.
• the method of the invention should allow more tumor toxicity at the same drug concentration, thus allowing for higher tumor doses without increasing toxicity to normal cells. Further, chemotherapeutic treatment of systemic tumor populations may also be improved by using the method of the present invention because lower doses of the drug may be possible by virtue of increased efficiency. Furthermore, local activation of the chemotherapeutic agent provides another benefit. Some chemotherapeutic agents require activation or conversion to their active state in cells or organs in the periphery, however, often the active (cytotoxic) metabolites cannot cross the blood brain barrier, and thus are not effective against brain tumors.
• the method of the invention should allow treatment of brain tumors by these chemotherapeutic agents.
• One such chemotherapeutic agent is CPA.
• CPA is largely ineffective against central nervous system neoplasms as its conversion to DNA-alkylating, cytotoxic metabolites is restricted primarily to the liver and these metabolites do not readily cross the blood-brain barrier.
• the use of the viral mutant of the invention, engineered to carry a cytochrome P450 gene and applied to a brain tumor would provide for local activation of CPA.
• a cytochrome P450 gene is utilized to sensitize central nervous tumor cells to the cytotoxic effects of cyclophosphamide (CPA).
• the transgene can also encode a cytokine to stimulate or enhance a tumor-directed immune response.
• cytokine to stimulate or enhance a tumor-directed immune response.
• TNF- ⁇ tumor necrosis factor alpha
• IL-2, IL-4 interferon- ⁇
• GM-CSF granulocyte-macrophage colony stimulating factor
• the transgene could also encode a tumor suppressor gene, or any other tumoricidal gene known to those skilled in the art, such as diptheria toxin (Coil- Fresno, P.M.. et al, Oncogene 74:243-247 (1997)), pseudomonas toxin, anti- angiogenesis genes, tumor vaccination genes, radiosensitivity genes, antisense
• RNA Ribonucleic acid
• ribozymes Zaia, J.A., et al, Ann. N Y. Acad. Sci. 660:95-106 (1992)).
• the herpes viral mutant with a tumor-specific or cell-specific promoter driving expression of the ⁇ 34.5 gene, further comprises a transgene encoding a gene product capable of converting a chemotherapeutic agent to its cytotoxic form or any other tumoricidal transgene, as mentioned above.
• the transgene can be inserted in the viral genome in any location where it will be expressed. Preferred locations in the viral genome for the transgene are at the locus of the original ⁇ 34.5 deletion or anywhere in the herpes UL40 locus.
• Exemplary candidates for treatment according to the presently claimed methods include, but are not limited to: (i) non-human animals suffering from neoplasms characterized by a tumor-specific promoter or cell-type specific promoter; (ii) humans suffering from neoplasms characterized by a tumor-specific promoter or cell-type specific promoter; (iii) humans or non-human animals in need of eradication of a particular cell population.
• neoplastic cells are intended cells whose normal growth control mechanisms are disrupted (typically by accumulated genetic mutations), thereby providing the potential for uncontrolled proliferation.
• the term is intended to include both benign and malignant neoplastic cells in both the central nervous system and the periphery.
• peripheral is intended to mean all other parts of the body outside of the brain or spinal cord.
• neoplastic cells include cells of tumors, neoplasms, carcinomas, sarcomas, papillomas, leukemias, lymphomas, and the like. Of particular interest are solid tumors that may arise in any organ or tissue of the mammalian body.
• Malignant brain tumors include astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma, Schwannoma, neurofibrosarcoma, and medulloblastoma.
• the treatment will be initiated by direct intraneoplastic inoculation.
• MRI, CT, or other imaging guided stereotactic techniques may be used to direct viral inoculation, or virus will be inoculated at the time of craniotomy.
• the vector would be inoculated into the tissue of interest.
• methods are known in the art for viral infection of the cells of interest.
• the viral mutant can be injected into the host at or near the site of neoplastic growth, or administered by intravascular inoculation.
• the viral mutant would be prepared as an injectable, either as a liquid solution or a suspension; a solid form suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
• the preparation also may be emulsified.
• the active ingredient is preferably mixed with an excipient which is pharmaceutically-acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
• the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH -buffering agents, adjuvants or immunopotentiators which enhance the effectiveness of the viral mutant (See, Remington 's Pharmaceutical Sciences, Gennaro, A.R. et al. , eds., Mack Publishing Co., pub., 18th ed., 1990).
• auxiliary substances such as wetting or emulsifying agents, pH -buffering agents, adjuvants or immunopotentiators which enhance the effectiveness of the viral mutant
• non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
• Aqueous carriers include water, aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
• Intravenous vehicles include fluid and nutrient replenishers. Determining the pH and exact concentration of the various components of the pharmaceutical composition is routine and within the knowledge of one of ordinary skill in the art (See Goodman and Gilman 's The Pharmacological Basis or Therapeutics, Gilman, A.G. etal, eds., Pergamon Press, pub., 8th ed., 1990). Additional formulations which are suitable include oral formulations. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Oral compositions may take the form of tablets, pills, capsules, sustained release formulations or powders and contain 10%-95%> of active ingredient, preferably 25-70%.
• the dosage of the viral mutant to be administered depends on the subject to be treated, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. For the most part, the virus is provided in a therapeutically effective amount to infect and kill target cells.
• the present invention also provides a method for selectively killing neoplastic cells that overexpress a known tumor-specific promoter using the herpes viral mutants described above, comprising: infecting said neoplastic cells with said herpes viral mutant, said viral mutant comprising: (a) a deletion or inactivating mutation in a gene encoding ⁇ 34.5; and (b) an insertion of at least one copy of said ⁇ 34.5 gene under the transcriptional control of said tumor- specific promoter, such that said promoter drives expression of said ⁇ 34.5 gene; and selectively killing said neoplastic cells.
• herpes viral mutant used in the above method, there may be more than one specific endogenous deletion or inactivating mutation of a herpes viral gene, in addition to the ⁇ 34.5 gene.
• the gene encoding RR encodes the small subunit.
• Any other herpes viral genes may also be deleted, such as, e.g. , thymidine kinase (TK), uracil DNA glycosylase (UNG), or dUTPase.
• TK thymidine kinase
• UNG uracil DNA glycosylase
• dUTPase dUTPase.
• Exemplary tumor-specific promoters are described above, and include, for example, CEA, AFP, tyrosinase, and PSA.
• Tumor cells are also known to overexpress particular oncogenes, so that cells with upregulated gene expression can be targeted using promoter elements of such genes.
• B-myb, C-myb, c-myc, c-kit, and the c-erbB2 oncogene are some representative examples of these types.
• the B-myb promoter (see, Lyon, J., et al, Crit. Rev.Oncogenesis 5:373-388 (1994) contains a consensus E2F binding site, is strictly regulated in cycling cells, and is in fact repressed in G 0 (Lam, E.W.
• the B-myb promoter is a particularly preferred tumor-specific promoter.
• the viral mutant used in the method is Myb34.5.
• Any cancer type having a well-characterized promoter would find use in the method of the invention.
• Examples of such promoters can be found in Table 1 of Clary, B.M., et al. , Cancer Gene Therapy 7:565-574 (1998); Table I of Spear, M.A., Anticancer Research 75:3223-3232(1998); Table 2 of Walther, W. and Stein, U., J.Mol.Med.74:379-392(1996); and Dachs, G.U., et al, Oncol. Res.
• the invention provides the above method for selectively killing neoplastic cells, wherein said herpes viral mutant further comprises a transgene, wherein the transgene is a suicide gene, a cytokine gene, or any tumoricidal gene. If the transgene is a suicide gene, then the method further comprises contacting the neoplastic cells with a chemotherapeutic agent capable of being activated by said suicide gene and selectively killing the neoplastic cells.
• the preferred suicide gene is cytochrome P450. P4502B1 is particularly preferred.
• the cytochrome P450 encoded is P450 2B6, P450 2A6, P450 2C6, P450 2C8, P450 2C9, P450 2C11, or P450 3A4.
• the chemotherapeutic agent is preferably a member of the oxazosphorine class, particularly cyclophosphamide, ifosfamide, N-methyl cyclophosphamide, methylchloropropylnitrosourea, polymeric cyclophosphamide, polymeric ifosfamide, polymeric N-methyl cyclophosphamide, or polymeric methylchloropropylnitrosourea.
• compositions containing the foregoing viral mutants wherein this composition may also contain one or more pharmaceutically acceptable excipients.
• Another embodiment of the present invention is a method for selectively eliminating a target cell population that overexpresses a known cell-specific promoter using the herpes viral mutants of the invention, comprising: infecting said target cells with said herpes viral mutant, said viral mutant comprising: (a) a deletion or inactivating mutation in a gene encoding ⁇ 34.5; and (b) an insertion of at least one copy of said ⁇ 34.5 gene under the transcriptional control of said cell-specific promoter, such that said promoter drives expression of said ⁇ 34.5 gene; and selectively eliminating a target cell population.
• selectively eliminating a target cell population is intended to include a significant reduction in the number of target cells versus non-target cells, as well as the complete or near complete elimination of target cells.
• herpes viral mutant used in the above method, there may be more than one specific endogenous deletion or inactivating mutation of a herpes viral gene, in addition to the ⁇ 34.5 gene.
• the gene encoding RR encodes the small subunit.
• Any other herpes viral genes may also be deleted, such as, e.g., thymidine kinase (TK), uracil DNA glycosylase (UNG), or dUTPase.
• Exemplary cell-specific promoters include the following: vascular endothelial growth factor (VEGF) receptor (flkl) promoter expressed in endothelial cells (Kappel et al Blood 93: 4282-4292 (1999); insulin promoter expressed in beta cells of the pancreas (Ray et al, J. Surg. Res. 84: 199-203 (1999); gonadotropin-releasing hormone receptor gene expressed in cells of the hypothalamus (Albarracin et al, Endocrinology 140: 2415-2421 (1999); matrix metalloproteinase 9 promoter, expressed in osteoclasts and keratinocytes (Munant et al, J. Biol. Chem.
• VEGF vascular endothelial growth factor
• flkl vascular endothelial growth factor receptor
• Examplary applications of this embodiment include the following: 1 ) Treatment options to eliminate a noxious cell population: For example, in conditions where there is exuberant neovascularization of blood vessels, such as cerebral Moya-Moya disease, use of the flk 1 receptor promoter to drive gamma
• 34.5 gene expression would allow for selective elimination of the blood vessels causing this disease.
• Another example is in conditions where there is extensive bone remodeling and elimination of bone, such as osteoporosis, the use of the matrix metalloproteinase 9 or the parathyroid hormone receptor to drive expression of gamma 34.5 would eliminate bone osteoclasts from further remodeling of bone.
• HSV1 herpes simplex virus
• Plasmids and Viruses - HSV strain F wild-type was acquired through the ATCC (Manassas,VA). Mutant virus R3616 (Chou, J., et al, Science 250:1262-1266 (1990)) (containing lOOObp BstEII-StuI deletions within both ⁇ 34.5 loci) was kindly provided by Dr. B. Roizman, University of Chicago.
• Mutant virus hrR3 (Goldstein, D.J. and Weiner, S.K., J. Virol. 62:196-205 (1988)) (kindly provided by S. Weller, University of Connecticut) contains an E. Coli lacZ cDNA inserted into the UL39 locus.
• the mutant virus MGH1 is characterized by insertion of the Escherichia coli lacZ cDNA into the UL39 locus and deletions of both ⁇ 34.5 loci, and it was constructed by recombination of the
• ICP6-lacZ region of hrR3 into the viral mutant R3616 (Kramm, CM., etal, Hum. Gene Ther. 5:2057-2068 (1997)).
• Plasmid pKX-BG3 which contains the lacZ gene within a 2.3 kb Xhol region of ICP6 (KOS origin, see, Goldstein,D.J. and Weller, S.K., J. Virol. 62:2970-2977 (1988)), was provided by S. Weller, as was plasmid pKpX2, which contains 2.3 kb of the ICP6 (UL39) gene.
• Plasmid pBGL34.5 containing the entire ⁇ 34.5 coding sequence, was provided by Xandra Breakefield and Peter Pechan (MGH).
• the B-myb promoter was excised as a Kpnl-Hindlll fragment from plasmid pBGL2myb (kindly provided by Dr. R. Watson, Ludwig Institute for Cancer Research, UK) and directionally cloned upstream of ⁇ 34.5.
• the plasmid used for the engineering of Myb34.5 by homologous recombination into MGH1 was designed to replace the lacZ cDNA in MGH1 in its entirety and delete an additional 888 nucleotides of ICP6 (UL39) sequence.
• the recombining plasmid (pKpX2-myb34.5) was engineered as follows. The full- length ⁇ 34.5 cDNA was excised as anNcol-Sacl fragment from pBGL34.5, it was blunt-ended, and then it was subcloned into pBSKII (Stratagene, La Jolla, Calif.) to generate plasmid pBS34.5.
• the B-myb promoter was excised as a Kpnl- Hmdlll fragment from pBGL2myb and directionally cloned upstream of ⁇ 34.5 in pBS34.5.
• the resulting plasmid, pKpX2-myb34.5 was then linearized with Seal and contransfected with MGH 1 viral D ⁇ A into Vero cells at various molar ratios with Lipofectamine (Gibco, Gaithersburg, MD). Virus progeny was harvested 5 to 7 days following transfection when cytopathic effects were evident. This progeny was released from cells through three cycles of freeze-thawing, and it was then plated onto a monolayer of Vero cells. After overlayering the monolayer with agarose, incubation at 37° C in an atmosphere containing 5%> carbon dioxide was performed.
• Plaques were then stained with 5-bromo-4-chloro-3-indolyl- ⁇ -D- galactopyranoside (X-Gal). Colorless plaques were selected as potential recombinants. These isolates underwent three rounds of plaque purification before having their genetic identity tested by Southern blot analysis.
• a Myb34.5 revertant (MybRevt) was engineered by using Myb34.5 as the parental strain and pKX-BG3 as the plasmid for homoglous recombination of the lacZ cD ⁇ A back into the ICP6 locus and deletion of the B-myb/y34.5 expression cassette.
• Probe labeling and hybridizations were performed using the ECL chemiluminescence system (Amersham) according to the manufacturer's protocol.
• Cell culture studies - All cells were cultured at 37 °C in an atmosphere containing 5% carbon dioxide in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal calf serum, 100U of penicillin/ml, and 10 ⁇ g of streptomycin/ml.
• DMEM Dulbecco's minimal essential medium
• Host protein synthesis shutoff studies were performed by infecting cells with viral strains for 16 hours. Cells were then placed in methionine-free medium for 10 minutes, then labeled using 35 [S]-methionine (New England Nuclear, Boston MA) for 90 minutes.
• Human glioblastoma cell lines U87, U373, T98G, and U343; rat gliosarcoma 9L cells; human neuroblastoma SKNSH cells and Vero (African green monkey) cells were obtained from the American Type Culture Collection
• mice Animal studies - Nude (nu nu) mice were obtained from the Cox 7 breeding facility, Massachusetts General Hospital (MGH). BALB/C mice were obtained from Charles River Laboratories (Wilmington, MA). Subcutaneous tumors were obtained by injection of 2 x 10 5 cells in the flanks of athymic mice (five animals per group for 9L gliosarcoma cells, and six animals per group for human U87 ⁇ EGFR glioma cells). Fourteen (for 9L) or ten (for U87 ⁇ EGFR) days after tumor implantation, animals with similar tumor volumes were randomly divided, and various viral strains were injected intratumorally at 5 x 10 7 PFU/dose in 100 ul volumes on days 1, 3, 5, and 7.
• mice were euthanized at day 33 (9L) or day 34(U87 ⁇ EGFR). Tumor volumes were measured with external calipers, as previously described (Wei, M.X., et al, Hum. Gene Ther. 5:969-978 (1994)).
• For neuro toxicity experiments BALB/C mice were stereotactically injected in the right frontal lobe (depth 3mm) with 10 ⁇ l volumes of virus at different dilutions, up to the highest stock titers obtainable. Animals were checked daily for 28 days. All animal studies were performed in accordance with guidelines issued by the MGH Subcommittee on Animal Care. Viral inoculation and care of animals harboring viruses were performed in approved viral vector rooms.
• Myb34.5 Genetic engineering of Myb34.5 -
• the multiply mutated virus Myb34.5 was constructed by recombining a B-myb promoter/ ⁇ 34.5 construct into the UL39 (also known as ICP6 or RR) locus of MGH1.
• MGH1 (Kramm, CM., et al, Hum. Gene Ther. 5:2057-2068 ( 1997)) was generated by recombining a lacZ cDNA into the ICP6 locus of the ⁇ 34.5 deletion mutant R3616 (Chou, J., et al, Science 250:1262-1266 (1990)).
• Figure IA provides a schematic of the DNA structure of Myb34.5.
• Fig. IB lacZ sequence
• Fig. IC lacZ sequence
• the parental virus, MGH1 contained a 9.0 kb Xhol l ?6-lacZ fragment (Kramm, CM., et al, supra.) that hybridized to an ICP6 probe (Fig. IB).
• Homologous recombination led to the deletion of lacZ and additional ICP6 sequence and insertion of the B-myb promoter/ ⁇ 34.5 sequence. This is evident by hybridization of the ICP6 probe to a 6.7 kb fragment in Myb34.5 DNA ( Figure 1 B).
• Hybridization with a lacZ probe revealed the absence of hybridizing fragments in digested DNA from Myb34.5 and the presence of the expected 9.0 kb hybridizing fragment in digested DNA from MGH1 (Fig. IC).
• BamHI-digested viral DNA was hybridized with a BstEl 1 -Bbs ⁇ 34.5 fragment (internal to the deleted regions). This demonstrated a ladder of hybridizing bands that is typically observed with the wild-type F strain. As discussed in the work of Chou et al.
• a revertant (marker-rescued) virus was also engineered, and designated MybRevt. This was achieved by homologous recombination, with Myb34.5 as the parental strain, and linearized pKX2-BG3 as the recombining plasmid.
• This plasmid contains the lacZ/ICP6 insertion and was used to create hrR3, the source of the ICP6::lacZ fusion region in MGH1 (Kramm, CM., et al, Hum. Gene Ther. 5:2057-2068 (1997); Goldstein, D.J. &
• the MybRevt revertant demonstrated a pattern on Southern hybridization to the ICP6 (Fig. IB), lacZ (Fig. 1 C), and ⁇ 34.5 (Fig. 1 D) probes, that was identical to that shown by MGH1 , the parent strain of Myb34.5.
• Functional expression of ⁇ 34.5 - To confirm that Myb34.5 produced functional ⁇ 34.5 protein, human SKNSH neuroblastoma cells were infected with a variety of viral strains.
• FIG. 2 shows that, as expected, MGH1 and the revertant virus (MybRevt) failed to prevent the infected cell response consisting of shut-off of protein synthesis, which is characteristic of intact ⁇ 34.5 function (Chou, J., 1992, supra). However, Myb34.5 and other strains with intact ⁇ 34.5
• wild-type F and hrR3 prevented the infected cell response (shut-off of protein synthesis), thus leading to viral protein production.
• Myb34.5 expressed functional ⁇ 34.5 protein was provided by assessment of viral replication in the U373 glioblastoma cell line which has been previously noted to restrict replication of ⁇ 34.5 mutant HSV strains (Mohr. I. and Bluzman, Y., EMBO J. 75:4759-5766 (1996)). After infecting 5 x 10 5 cells at a MOI of 1.0 and harvesting viral output 48 hours later, Myb34.5 yields were similar to those of wild-type F strain (1.1 x 10 7 PFU vs. 5.0 x 10 7 PFU, respectively).
• Murine fetal striatal neurons were infected, and viral yields were assessed by plaque assay on Vero cells (which do not require ⁇ 34.5 for efficient viral replication). While the wild-type F strain demonstrated vigorous replication in neurons, all the mutant strains, including Myb34.5, demonstrated minimal viral replication (See, Table 1).
• Myb34.5 demonstrated greater oncolytic efficiency in vitro than did the the parental strain MGHl , and for some tumor cell lines its oncolytic efficacy approached that of the wild-type virus (Table 3). These findings thus showed that the oncolytic effect of Myb34.5 was greater than that of the ⁇ 34.5 mutant viruses (MGH 1 , MybRevt, and R3616, whose killing efficacy closely replicates that of MGHl).
• MGHl and other ⁇ 34.5 mutants may have limited efficacy due to the lower viral yields obtained from infected cells (Kramm, CM., et al, supra).
• Myb34.5 like MGHl or other ⁇ 34.5 mutants, demonstrated little pathogenicity in mice at intracerebral doses of 10 7 PFU, remaining neuroattenuated. Therefore, Myb34.5 exhibits improved oncolytic efficacy compared to the parental mutant MGH 1 , the marker-rescued revertant MybRevt, and MGHl's parental mutant R3616, while maintaining characteristics of neuroattenuation, with an LD 50 of > 10 7 PFU. This value is qualitatively similar to those observed with ⁇ 34.5 mutants, although strict quantitative comparisons were limited by the technical inability to accurately determine an LD 50 for the latter group of mutants .
• a major objective in cancer gene therapy is to identify viral mutants that provide significant anticancer effects while at the same time demonstrating minimal side effects and toxicity towards normal cells and tissues.
• This feat has been accomplished by engineering replication-defective viral vectors, which should display minimal toxicity toward infected normal and tumor cells, and endowing them with the ability to express anticancer genes to achieve biologic effects (Moriuchi, S., et ⁇ /., Cancer Res. 55:5731-5737 (1998)).
• Such vectors When applied as inocula to large human tumor masses, such vectors do not diffuse well due to their size, thus limiting anticancer effects to cells located in proximity to the injection tract (Bobo, R.H., et al, Proc. Natl. Acad. Sci. USA 97:2076-2080 (1994);
• One potential solution to this problem consists of the use of replication- conditional (oncolytic, replication-restricted) viral mutants that maintain the ability to replicate in a relatively selective fashion in tumor or mitotic cells while being restricted in their ability to replicate in normal cells.
• Such viral mutants would thus propagate from initially infected tumor cells to surrounding tumor cells, thus achieving a larger volume of distribution and enhanced anticancer effects.
• Myb34.5 offers the theoretical advantage of being less prone to recombinatorial repair to wild-type in the presence of latent pre-existing, or subsequent HSV infection.
• RNA-dependent kinase phosphorylates the alpha subunit of elongation initiation factor 2, resulting in inhibition of protein synthesis (Chou, J., et al, Science 250:1262- 1266 (1990); Chou, J., et al, J. Virol. 65:8304-8311 (1994); Chou, J. and Roizman, B., Proc. Natl Acad. Sci. USA 59:3266-3270 (1992)).
• Infection of cells of neuronal origin with mutants incapable of expressing ⁇ 34.5 results in shutoff of cellular protein synthesis, with the resultant limitation of viral production.
• Myb34.5 when cells were serum stimulated, titers of Myb34.5 increased by 3 orders of magnitude, approaching titers observed with strains F and hrR3, while titers of the ⁇ 34.5 mutants (MGHl and MybRevt) increased only slightly. It was notable that the basal level of replication of Myb34.5 was lower than that of hrR3 in quiescent cells and was more quantitatively similar to that of the RR ⁇ 34.5 mutant MGHl . Myb34.5 demonstrated a higherfold induction of replication in cycling cells than hrR3 , while MGH 1 and MybRevt showed minimal induction, suggesting that the effects of the Myb34.5 construct exceed the effect of simple complementation of ribonucleotide reductase.
• Myb34.5 would thus replicate at relatively low levels (similar to the levels observed with MGHl) in cells that are quiescent, but infection of dividing brain tumor cells would produce a significant increase in viral titers, thus providing a therapeutic advantage over MGHl or other ⁇ 34.5 mutants.
• Infection of normal brain cells can occur with ⁇ 34.5 mutants (Kesari, S., et al, J. Gen. Virol 79:525-536 (1998); Kesari, S., et al, J. Neurosci. 76:5644- 5653 (1996); Markovitz, N.S., et al, J. Virol.
• Myb34.5 may also provide a suitable backbone for the addition of anticancer genes, such as those that activate prodrugs.
• anticancer genes such as those that activate prodrugs.
• tumor-selective promoters it would be relatively easy to use these to control expression of the ⁇ 34.5 gene or other virulence genes in order to further restrict viral production to tumor versus normal cells.
• the approach described here may also be used to restrict virulence to specific cell types in a tissue, by employing cell-specific promoters.
• the B-myb promoter contains a consensus E2F binding site, is strictly regulated in cycling cells, and is in fact repressed in G 0 (Lam, E.W. and Watson, R.J., EMBO J. 72:2705-2713 (1993); Lam, E.W., et al, Gene 760:277-281 (1995); Bennett, J.D., et al, Oncogene 73: 1073-1082 (1996)).
• a replicafion- defective adenovirus containing an E2F-responsive promoter has been used to demonstrate tumor-specific gene expression, relative not only to quiescent neuronal tissue but also to nontransformed normal cycling cells (Parr, M. J., et al, Nat. Med. 3:1145-1149 (1997)).
• the primary difference from the strategy described in the present report is the use of a promoter that may be considered tumor or cell cycle specific instead of hepatocyte specific, and the use of an HSV gene that is directly related to virulence ( ⁇ 34.5) rather than an essential transcription factor, such as ICP4.
• a promoter that may be considered tumor or cell cycle specific instead of hepatocyte specific
• HSV gene that is directly related to virulence ( ⁇ 34.5) rather than an essential transcription factor, such as ICP4.
• Myb34.5 the exemplified virus, designated Myb34.5, is replication competent, and targeted virulence is obtained by regulating viral replication and direct oncolysis.
• Myb34.5 represents a novel, targeted oncolytic herpesvirus and adds to two recently described tumor-selective, oncolytic adeno- and reoviruses (Bischoff, J.R., et al, Science 274:373-376 (1996); Coffey, M.C, et al, Science 252:1332-1334 (1998)).
• the E IB-defective adenovirus ONYX-015 is thought to depend on alterations of the p53 tumor suppressor pathway for efficient replication in tumor cells,although this mechanism has recently been called into question (Goodrum, F.O., et al, J. Virol. 72:9479-9490 (1998)).
• Myb34.5 may take advantage of alterations of the pl6/cdk4/RB/E2F pathway, and adds to the possibility that multiple tumor genetic alterations may be targeted by different viral treatment strategies.
• the strategy of using cell-specific or tumor-specific promoters to drive expression of the ⁇ 34.5 gene may also be suitable as a means to eliminate selected cell populations in vivo.

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